Human Aldo-Keto Reductases (AKRs) involved in steroid hormone transformation include AKR1C1 AKR1C4, which display different ratios of 3a-17beta- and 20a-hydroxysteroid dehydrogenase (HSD) activity, md AKR1D1 which is steroid 5b-reductase. The HSDs catalyze the formation and elimination of potent androgens, estrogens, and progestins and may regulate ligand occupancy and trans-activation of steroid hormone receptors in target cells. Inhibitors of these enzymes could result in tissue specific hormone responses and would belong to a new class of therapeutics called selective intracrine modulators (SIMs). To aid SIM design, extensive structure-function studies (x-ray crystallography and site-directed mutagenesis) have been performed on rat 3a -HSD (AKR1C9), which is the most thoroughly characterized HSD. However, significant unanswered questions remain. The complete kinetic mechanism, identity of the rate-determining step, and identity of the transition-state is lacking for any steroid hormone transforming AKR. In Aim#l, stopped-flow spectroscopic methods will determine whether cofactor binding or release, steroid binding or release, or turnover are rate-limiting in AKR1C9. Primary and solvent kinetic isotope effect measurements will determine whether hydride transfer or proton donation is rate-controlling in the chemical step and will lead to the identification of the transition-state. In Aim#2, we will test the structural basis of catalysis and steroid hormone recognition in human AKRs. Site-directed mutagenesis will validate whether a point mutation El20 in the catalytic tetrad of 5b -reductase is responsible for steroid double-bond reduction. The crystal structure of human type 3 3a-HSD(AKR1C2)[unreadable]NADP+[unreadable]oursodeoxycholate complex will guide site-directed mutagenesis to test the roles of individual amino acids in binding bile-acids (inhibitors) with nanomolar affinity; and pocket residues that differ in this structure from other AKR1C isoforms will be mutated to determine whether the ratios of 3a - 17b - and 20a -HSD activities can be altered. In Aim#3, we will use transient kinetic approaches to determine why the human enzymes have such low k values and where the reaction becomes "stalled". In Aim#4 the xray crystal structures of AKR1C8 and AKRIC1 (rat and human 20a -HSD) complexed with NADP+ and progesterone are sought to determine how AKRs catalyze the 20a -HSD reaction, previous crystal structures have provided a structural basis for the 3a- and 17b -HSD reactions only. Completion of these aims will provide structural details of how AKRs transform steroid hormones.